Dr. Weinmaster's working hypothesis is that genes like Drosophila Notch, mediate a subset of cell-cell interactions that help specify cell fate. The specific aims are designed to provide information required to reveal the role that Notch genes play during mammalian development. Using in situ hybridization and immunohistochemical techniques she will determine the cell types and the developmental processes in which these two closely related genes are expressed. Information provided by these studies will identify candidate cell-cell interactions mediated by the Notch genes, which could potentially be used as cell culture systems to measure Notch activity. The question of whether Notch mediates cell-cell interactions will be studied using neuron-Schwann cell cocultures. If Notch mediates interactions between neurons and Schwann cells then genetically engineered Notch-minus Schwann cells will be unable to respond to neuronal contact. Constitutive expression of Notch in transfected C2C12 myoblasts prevents myotube formation, providing a convenient "read-out" of Notch activity. Dr. Weinmaster will use this assay to determine the structural motifs of Notch that are required to inhibit myogenic differentiation of C2C12 cells and thereby begin to investigate the mechanisms of Notch function. An important complement to these cell culture experiments would be the isolation of ligands for Notch. Cells expressing either Notch or its ligand could then be cocultured and the downstream effects of cell-cell interactions, mediated through Notch, would be monitored to determine the events required for Notch- signal transduction. The ultimate goal of this work is to define the function and mode of action of Notch1 and Notch2 in mammalian cells during normal development.